Yersinia pestis is the causative agent of plague, a pandemic clinically divided into bubonic, septicemic, and pneumonic diseases. However, such empiric distinctions oversimplify the interactions and progression among these clinical presentations, particularly for the type of acute lung injury (ALI) and septic shock originating as pneumonic plague that will dominate epidemics following inhalation of Y. pestis as a weapon of bioterrorism. To identify the molecular pathogenesis of Y. pestis-induced shock and ALI, we will evaluate the relative importance of virulence factors that collectively promote macrophage adherence and phagocytosis, but blunt innate antimicrobial responses despite concomitant signaling by Y. pestis endotoxin. It is unclear to what extent cytokines modulate the severity of ALI after lower respiratory tract Y. pestis infection (perhaps with minimal localized proinflammatory cytokine expression), or its progression to septic shock with disseminated intravascular coagulation (DIG) and multiple organ failure (MOF). Yersinia's survival in blood requires chromosomal factors including the pigmentation (pgm) locus; pgm strains are avirulent by peripheral route, but at 37 degrees C can still suppress macrophage function and cause disease. Yersinia also initially 'tranquilizes' leukocytes by a low-calcium-response (Icr) plasmid encoding Yop effector proteins. Thus, Icr Y. pestis are even less virulent than pgm- strains due to prompt leukocyte recognition and curative cytokine secretion. In March 2003, the CDC excluded from its Select Agents and Toxins Class A List several Icr variants of established wild type (wt) strains such as KIM6 (1961 medievalis biotype) and CO92 (1992 orientalis biotype, since renamed C099), and then in May 2003 excluded pgm variants as well. In April 2003, we obtained Icr KIM6 for preliminary studies from Dr. Sue C. Straley (U. Kentucky), who will also provide the pgm CO99 and WT/CO99 to be used here. From our preliminary data, we hypothesize that distinct proinflammatory and antimicrobial host responses to pgm- CO99 vs. wt/CO99 infections will determine the severity of ensuing ALI and septicemic disease, due to critical differences in bacterial pathogenesis and in the efficacy of these host responses. We propose four Specific Aims. 1) Establish the time courses and mechanisms of pneumonic plague-induced ALI and disease progression in conscious rats infected intratracheally (i.t.) or by bioaerosol nebulizer with graded inocula of pgm- CO99. Instrumented rats will be evaluated for evolving ALI, cardiovascular dysfunction, hematology, and serum cytokines to identify those i.t. and aerosol inocula that cause ALI, disseminated infection, and septic shock within 2-4 d of bacterial infection. 2) In animals infected i.t. or by aerosol with Aim 1-refined inocula of pgm- CO99, determine changes in systemic vascular resistance, organ function and coagulation status, as well as the bacterial burden, vascular permeability, histopathology, and cytokine gene expression in lung, liver, heart, and spleen. [Aims 1 and 2 to be completed in Year 1 within BSL-2 labs.] 3) Compare key findings of pgm- CO99 infection with results for naive rats infected i.t. or by aerosol with WICO99. Disease in such animals should closely resemble human pneumonic plague leading to ALI, disseminated disease, and septic shock within 2-4 d of bacterial challenge and at smaller inocula than with pgm- CO99. 4) Determine the relative efficacy of 2 novel therapeutic strategies employing antimicrobial and anti-inflammatory approaches i). nebulizer-aerosol vs. intravenous gentamicin antimicrobial therapy initiated early (< 24 h post-infection) or later (" 48 h) to rats aerosol-infected with wtCO99; and ii). antimicrobial therapy with or without simultaneous i.v. treatment with recombinant activated protein C to limit acute inflammatory responses predisposing to ALI , DIG, and progression to MOF and otherwise fatal septic shock. [Aims 3 and 4 to be completed in Year 2 within BSL-3 labs.]